Protein structure determination is a key step in developing molecular-level understandings of the role of proteins in cell signaling pathways, which in turn can guide understanding of diseases and the rational design of potential drugs for treatment. High-resolution structures of relatively large proteins are generally obtained by X-ray diffraction from protein crystals. Consequently, identification of the conditions amenable to the formation of diffraction-quality protein crystals remains a major bottleneck in the sequence to structure pipeline. Because of the large chemical space associated with finding the appropriate crystallization conditions, it is routine to perform hundreds of crystallization trials for a given protein target. High throughput approaches for rapidly preparing and screening large numbers of crystallization trials have improved the pace of structure-discovery, increasingly placing the bottleneck for protein structure determination on the development of reliable and automated methods for protein crystal detection for efficiently mapping and sampling chemical space.
Numerous strategies have been adopted for selectively and rapidly identifying protein crystals. The simplest experimental approaches rely on visual inspection or algorithmic analysis of bright-field images, which can be error-prone (high false-positive/false negative rates) and particularly challenging for small (<5 μm) crystals. UV fluorescence of intrinsic aromatic residues is also widely used, offering improved image contrast and facile discrimination between protein crystals and small-molecule crystals of salts or other additives in the mother liquor. However, the deep uv excitation light used for intrinsic UV fluorescence exhibits poor transparency in conventional optical elements and most polymeric materials used for crystallization screenings, posing practical limitations on its general applicability. Furthermore, UV fluorescence cannot easily discriminate between disordered aggregates and microcrystalline conglomerates. Moreover, the high-energy photons used for UV-imaging (<280 nm) can induce photochemical damage to proteins during long or repeated exposures through the breakage of disulfide bonds and polymerization of neighboring residues within the crystalline lattice. The use of attenuated total reflection FT-IR spectroscopic imaging of protein crystallizations has recently been demonstrated as a technique that can distinguish between protein and precipitant crystals. Although this technique has the ability to selectively image protein crystals through the protein-specific amide bond at 1550 cm−1, it is difficult to implement on traditional crystallization screening platforms, such as 96 well plates, thus making it a limited and specialized technique. Other techniques for discriminating between protein and precipitant protein crystals are the “crush test” where the crystals are deemed protein if they disintegrate when touched with a needle and the staining of crystals with Coommasie Brilliant Blue dye, both of which are destructive techniques. More recently, second order nonlinear optical imaging of chiral crystals (SONICC) based on second harmonic generation (SHG) microscopy has been shown to be highly selective for protein crystal detection. Coherent SHG only arises from assemblies with long-range order, allowing selective identification of protein crystals with negligible contributions from solvated proteins or amorphous aggregates.
Although SONICC remains an attractive option, one alternative approach to shift the fluorescence of protein crystals to the visible region of the spectrum is to incorporate organic fluorophores into protein crystals at low doping densities by addition to the mother liquor. Although the degree of incorporation within the crystalline lattice can vary substantially depending on the nature of the fluorophore and the protein, the resulting fluorescence intensity of the crystal will often be higher than the surrounding mother liquor by nature of the higher local density. Furthermore, low doping densities have the advantage of suppressing autoquenching, which can substantially reduce the overall quantum efficiency for emission of intrinsic chromophores (e.g., tryptophan). A major disadvantage of fluorophore doping is that protein crystals can routinely exhibit low or moderate doping efficiencies, with the fluorescence from solvated dye increasing the background and reducing image contrast.
Detection of intrinsic visible light emission from native, unlabeled protein crystals can reduce complications associated with incorporation of large organic chromophores while still maintaining compatibility with conventional optical components and detectors. Intrinsic one-photon excited emission of visible light has been reported in previous studies of seemingly innocuous organic assemblies lacking obvious chromophores, including proteinacious material, triethylamine, poly(amido amine) dendrimers, aliphatic polyamides, poly(ether amide)s, poly(propylene imine), and poly(amine-amide)s. In those studies, a combination of fluorescence and phosphorescence was observed, with oxygen implicated as a critical component, possibly through oxygen exiplex formation with amino groups. This mechanism suggests that visible emission could potentially be observed in broad classes of organic species.